Immunoanalytical Techniques

Question 1

What is a hapten?

Answer 1

Small molecule that binds to an antibody but not capable producing response unless bound to macromolecule

Question 2

Explain the antibody structure

Answer 2

• Immunological G class

- 4 polypeptide chains held together by disulfide bonds

Question 3

Difference between monoclonal or polyclonal antibodies?

Answer 3

Monoclonal - same binding strengths and specificity

Polyclonal - different binding strengths and specificity

Question 4

Steps of production for Monoclonal antibodies? Polyclonal?

Answer 4

Monoclonal

1) Immunize animal and collect Beta lymphocytes from spleen
2) Fuse with myeloma cells to make Beta lymphocytes that produce antibodies
3) Screen non-antibodies by HAT
4) Culture and extract

Polyclonal

1) Inject immunogen into animal
2) Conduct 3 month booster shots
3) Collect serum

Question 5

What does antigen labeling help with?

Answer 5

Determining bound vs unbound antigen or antibodies

Question 6

What are the the types of assays used to determine bound vs free antigen? How does each one work?

Answer 6

Heterogeneous Immunoassay
- physically separate bound vs free by precipitation, adsorption, etc.)

Homogeneous Immunoassay

• uses properties of free and bound to separate
• free antigen is more water soluble
• bound antigen is more lipid soluble

Question 7

What are the requirements for labels in immunoassays?

Answer 7

• Needs to produce a high response
• Low Background
• Water soluble conjugates
• Negligible effect on Ab binding (dependent on size)

Question 8

What are some disadvantages of radioactive labels?

Answer 8

Poor shelf life
Health Hazard
Requires heterogeneous procedure

Question 9

What are advantages/limitations of enzyme labels?

Answer 9

Advantages

• Simple instrument for detection
• Long shelf life
• Homogeneous assay possible
• Enzyme multiplication possible bc enzyme is reusable

Limitations

• May affect Ab binding (large)
• special requirements for homogeneous assay
• control conditions are crucial (temp, time, etc.)

Question 10

What are advantages/limitations of fluorescent labels?

Answer 10

Advantages
- pretty much same with radioactive

Limitations

• non-selective label
• long wavelengths are needed for selectivity

Question 11

What are two techniques of solid phase methods?

Answer 11

Immunoabsorbent based method

Sandwich method

Question 12

What is a real life example of a heterogeneous immunoabsorbent solid phase method? How does it work?

Answer 12

Profile II is urine sampler that tests for illicit drug use. Antibodies bind to drug in urine and trickles down test strip. Unbound antibodies then attach to drugs at the end which produces a line.

Question 13

What is a real life example of a heterogeneous sandwich solid phase method? How does it work?

Answer 13

Pregnancy test
Tests for hcG (human chorionic gonadotropin). First line is where antibodies with hcG sandwich with other antibodies. Free antibodies then form a line at the end to act as a control.

Question 14

What are some errors that can occur with test kits?

Answer 14

• high temps denature antibody
• detergents denature antibody
• poor saturation of adsorbent
• physiological false positives and negatives

Question 15

What is an example of a homogeneous technique with enzymes? How does it work?

Answer 15

EMIT: Enzyme multiplied Immuno technique

The antigen from sample competitively binds to the enzyme against the labeled antigen

Question 16

What is an example of a homogeneous technique with fluorescence? How does it work?

Answer 16

Fluorescence Polarization Immunoassay (FPI)

• Antibody + antigen spins slower and retains polarization
• antigen by itself loses polarization if spun and will not fluoresce